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Can DNA enhance your look?

Editor's Introduction

Fine tuning of craniofacial morphology by distant-acting enhancers

We're all familiar with the adage that no two faces are alike. But, how is this tremendous amount of variation possible? Using genetic tools and three-dimensional imaging, this paper makes the case that subtle tweaks in non-protein-coding DNA influence the shape of the developing face.

Abstract

The shape of the human face and skull is largely genetically determined. However, the genomic basis of craniofacial morphology is incompletely understood and hypothesized to involve protein-coding genes, as well as gene regulatory sequences. We used a combination of epigenomic profiling, in vivo characterization of candidate enhancer sequences in transgenic mice, and targeted deletion experiments to examine the role of distant-acting enhancers in craniofacial development. We identified complex regulatory landscapes consisting of enhancers that drive spatially complex developmental expression patterns. Analysis of mouse lines in which individual craniofacial enhancers had been deleted revealed significant alterations of craniofacial shape, demonstrating the functional importance of enhancers in defining face and skull morphology. These results demonstrate that enhancers are involved in craniofacial development and suggest that enhancer sequence variation contributes to the diversity of human facial morphology.

Using what they learned from the reporter mice, the researchers chose 3 enhancers to delete and then measure the effect on adult mouse skull shape.

Enrichment analysis identified 4399 distal candidate enhancers genome-wide, defined as regions that showed significant p300 binding in craniofacial tissue and were at least 2.5 kb from known transcription start sites (Fig. 2and tables S1 and S2). Candidate enhancers were located up to 1.4 Mb (median distance, 44 kb) from the nearest known transcript start site, with 38.4% in introns of genes and 54.7% located in noncoding regions outside of genes (intergenic). The majority of candidate enhancers also showed evidence of evolutionary constraint (87.5%) (table S1) and had unique orthologous sequences in the human genome (96.7%). Unbiased ontology analysis (33) revealed that candidate craniofacial enhancers are enriched near genes that are known to cause craniofacial phenotypes when deleted in mouse models or mutated in humans (table 1). Candidate craniofacial enhancers were also enriched at loci implicated in human craniofacial traits and birth defects through genome-wide association studies (fig. S1). These observations are consistent with a role of the identified enhancer candidate sequences in the regulation of genes with known roles in craniofacial development. Taken together, these results suggest that thousands of distant-acting enhancers are involved in orchestrating the genome-wide gene expression landscape during craniofacial development.

Fig. 2. Genome-wide identification of candidate craniofacial enhancers. Mouse genome graph showing all p300-enriched regions (green dots) and all 281 sequences tested in vivo or reexamined for craniofacial activity in this study (red dots). Examples of selected major craniofacial genes (55) and genomic regions [such as regions orthologous to human 8q24 (43) and ABCA4(46)] are highlighted by pink boxes. Known craniofacial loci were generally enriched in candidate sequences and were specifically targeted for sampling in transgenic assays (red dots). The three genomic regions studied by means of knockout analysis are highlighted by blue boxes.

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This figure gives an overview of the mouse chromosomes, each represented by gray bars. The authors have overlain annotations of candidate enhancers, candidate enhancers where they visualized activity, and regions of the genome that are important in craniofacial development in mice or similar to human genome regions important in human craniofacial development.

Table 1. Top enriched annotations of mouse and human phenotypes associated with candidate craniofacial enhancers. (Top) Ten of the 12 most significantly enriched terms from the mouse phenotype ontology directly relate to craniofacial development. The remaining two phenotypes (abnormal axial skeleton morphology and abnormal skeleton development) relate to general skeleton development, a process that shares key signaling pathways with cranial skeleton development (58). (Bottom) Six of the 10 most significantly enriched terms from the human phenotype ontology are relevant to craniofacial development. The four remaining phenotypes are all associated with limb abnormalities, which is consistent with previous knowledge of shared developmental pathways during limb and face development (59–61). In each analysis, only terms exceeding twofold binomial enrichment were considered and ranked by P value (binomial raw Pvalues).

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Each of their putative enhancers was assigned to its likely target (a protein-coding gene) and then a gene database was searched for known functions and diseases associated with these genes. These are the top search terms that corresponded with their searches.

To illustrate the reproducibility and diversity of craniofacial activity patterns identified in transgenic embryos, selected examples of enhancers identified in this study are shown in Fig. 3A. For all craniofacial prominences (medial nasal, lateral nasal, maxillary, and mandibular), structure-specific active enhancers were identified (Fig. 3A; a schematic view of the e11.5 mouse face is provided in fig. S4A). In depth analysis of craniofacial activity patterns through the combined use of whole-mount LacZ staining and OPT imaging revealed that in many cases only subregions of these structures were reproducibly targeted by an enhancer. For example, enhancer mm387 drives expression in the anterior part of the maxillary prominence, whereas enhancer mm458 is restricted to a posterior ventral region (Fig. 3B, top). Similar region-specific activities are observed in other facial substructures—such as the nose, where enhancer mm933 is active in the medial nasal prominence, whereas the activity of enhancer mm426 is confined to the lateral nasal prominence (Fig. 3B, top). OPT scans of whole-mount embryos provide additional spatial information about enhancer activity pattern by capturing the activity signal in internal embryonic structures (Fig. 3B, bottom). These data highlight the complexity, diversity, and spatially highly restricted activity patterns of distant-acting enhancer sequences active during craniofacial development.

Fig. 4. Regulatory landscapes of craniofacial loci. (A) Craniofacial enhancers near Msx1, a major craniofacial gene, were identified with p300 ChIP-Seq (green boxes). This included the reidentification of a region proximal to Msx1 with previously described enhancer activity (mm426) (56), as well as four additional, more distal enhancers with complementary activity patterns. For each enhancer, only one representative embryo is shown; numbers indicate reproducibility. Red arrows indicate selected correlations between Msx1 RNA expression (ISH) and individual enhancers. Red box indicates enhancer hs746, which was further studied by means of knockout analysis. Msx1 ISH is from Embrys database (http://embrys.jp) (57). (B) Identification of craniofacial enhancers in the cleft- and morphology-associated gene desert at human chromosome 8q24 (orthologous mouse region shown) (43). Brown box indicates the region corresponding to a 640-kb human region associated with orofacial clefts [nonsyndromic cleft lip with or without cleft palate (NSCL/P)] and devoid of protein-coding genes. Two of four candidate enhancers within the region drove craniofacial expression. For each enhancer, lateral and frontal views of one representative embryo are shown. (C) Identification of a craniofacial midline enhancer at the cleft-associated susceptibility interval at the ABCA4 locus (46). The enhancer is highly active in the nasal prominences (yellow arrows), but not the maxillary or mandible (pink arrows). Embryos have an average crown-rump length of 6 mm.

Panel A

This figure shows where enhancers are located relative to the Msx1 gene.

Note that enhancer activity does not necessarily correspond with gene expression. Additionally, an enhancer is not necessarily driving expression of the nearest gene. Enhancer activity of mm429 does not match up as neatly with Msx1 expression as enhancers mm426 and hs746 do.

Panel B

Though it doesn't encode any proteins, genome studies have identified a region of the genome in which certain noncoding sequence changes are associated with an increased risk for nonsyndromic cleft lip and/or palate.

Interestingly, this paper's ChIP-Seq screen identified 4 candidate enhancers within this locus. Two of which demonstrate reproducible patterns of craniofacial activity.

Panel C

One enhancer, also found in a craniofacial clefting-associated locus, shows striking, strong activity along the midline. These LacZ stains and optical slices through the embryo illustrate the regions of enhancer activity.

Craniofacial Enhancers Within Disease-Associated Intervals

To illustrate the utility of these enhancer data sets in the follow-up of genome-wide association, population-scale sequencing, and candidate locus studies, 50 candidate enhancers mapping to intervals implicated in craniofacial morphology or orofacial birth defects through human genetic studies were included in the transgenic assays (table S3). Trait-associated variants that map to noncoding genome regions or are not linked to any protein-altering variants are a common challenge in the interpretation of such genetic studies. A prototypical example is a region of human chromosome 8q24 that is devoid of protein-coding genes. A 640-kb stretch located within this region is a major susceptibility locus for cleft palate, with a calculated population attributable risk of 41% (43–45). Variants at this locus are also significantly linked to normal variation in several facial morphology traits (16). We identified four craniofacial enhancer candidate sequences in the mouse genome region orthologous to the human risk interval, two of which drive reproducible craniofacial reporter activity at e11.5 in transgenic mice (Fig. 4B). As a second example, we examined the 1p22 locus. In this interval, markers located near and within the ABCA4 gene are associated with an increased risk for cleft palate in humans, but it remains unclear whether these variants are linked to deleterious protein-coding mutations of ABCA4 (46, 47). On the basis of RNA expression data, the neighboring gene ARHGAP29, rather than ABCA4 itself, has been proposed to be causatively involved in craniofacial development (48). However, ARHGAP29 falls outside the genomic boundaries of the risk-associated linkage block. By scanning the region comprising these two genes for possible associated enhancers, we identified a human-mouse conserved sequence in the first intron of Abca4 that drove highly-reproducible reporter activity in the facial midline, a pattern reminiscent of Arhgap29 RNA expression, suggesting that this enhancer may drive expression of Arhgap29 during craniofacial development (Fig. 4C and movie S10) (49). A causative effect of sequence or copy number variants in these particular enhancers on craniofacial morphology remains to be demonstrated; furthermore, we cannot exclude the existence of additional enhancer sequences at these loci that were not captured in the present screen. These possible limitations notwithstanding, our results illustrate the utility of collections of validated enhancers as starting points for the mechanistic interpretation of human genetic studies by linking functional genomic and human genetic data sets.

Targeted Deletions of Craniofacial Enhancers

The existence of large numbers of distant-acting enhancers with precise tissue-specific activities during craniofacial development raises the question of their functional impact on craniofacial morphology through the regulation of their respective target genes. To examine such contributions in more detail, we selected three enhancers with highly reproducible craniofacial activity patterns and explored their functions through targeted deletions in mice (Fig. 1). The three enhancers—termed hs1431 (near Snai2), hs746 (near Msx1), and hs586 (near Isl1)—were chosen on the basis of their association with known craniofacial genes (supplementary text) (7,50, 51), the robustness of their activity patterns, and the absence of additional known enhancers with overlapping activity near the same gene. Furthermore, the in vivo activity patterns driven by these enhancers partially recapitulate the known expression patterns of their presumptive target genes (Fig. 4A and fig. S3). The enhancers were intentionally chosen from different, functionally unrelated loci in order to provide a representative sample of the genome-wide enhancer data set, rather than an in-depth exploration of a single gene or pathway. All selected enhancers are located at a very long distance from their respective target genes (350, 235, and 190 kb, respectively) and are active in the craniofacial complex through multiple stages of embryonic development (Figs. 4A and 5, fig. S3, and movies S1 to S9).

Fig. 5. Developmental activity patterns of three enhancers selected for deletion studies. The in vivo activity of each enhancer was monitored at different stages of development (e11.5, e13.5, and e15.5) (movies S1 to S9). All enhancers were reproducibly active in the craniofacial complex during embryonic development, with spatial changes in activity across stages. Side views are of LacZ-stained whole-mount embryos. Front views are optical projection tomography reconstructed 3D images. Regions of enhancer activity are shown in red.

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As facial structures develop, and the embryo matures, we see enhancer activity become restricted to smaller and smaller regions.

The three enhancers in this figure are the ones they selected for their enhancer knockout experiment.

Panel A

This image shows hs1431 enhancer activity in structures of the developing face as well as the developing fore- and hindlimbs.

Panel B

This image shows hs746 enhancer activity in the developing eye and snout.

Panel C

This panel shows that when enhancer hs1431 is deleted in a developing embryo, Snai2 expression is significantly decreased throughout the developing craniofacial complex. These results are compatible with the pattern of hs1431 activity from figure 5.

These results suggest that hs1431 is in fact an enhancer responsible for promoting Snai2 expression in the developing face.

Panel D

When enhancer hs746 is deleted in a developing embryo, Msx1 expression is significantly decreased in the developing mandible and maxilla, but not in the nose. These results are compatible with the pattern of hs746 activity from figure 5.

These results suggest that hs746 is in fact an enhancer responsible for promoting Msx1 expression in the developing upper and lower jaws.

To examine whether the deletion of these enhancers altered craniofacial morphology, we compared mouse skulls from wild-type and enhancer deletion mice at 8 weeks of age. Because it is challenging to quantify possible differences in craniofacial morphology with visual observation alone, we used micro-computed tomography (micro-CT) to obtain accurate 3D measurements of the skulls. Three cohorts, each consisting of at least 30 mice homozygous for a deletion of one of the three enhancers, were compared with a cohort of 44 wild-type littermates. Micro-CT reconstructions of each mouse head were measured by using 54 standardized skeletal landmarks (fig. S5). The cohorts of wild-type and enhancer deletion mice were compared by using canonical variate analysis (CVA) to identify possible changes in craniofacial morphology resulting from the enhancer deletions (Fig. 7). Procrustes analysis of variance (ANOVA) (F = 12.0, P < 0.0001) and multivariate ANOVA (Pillau’s Trace 2.5, P < 0.0001) tests both showed that enhancer deletion genotypes were significantly associated with alterations of craniofacial shape. All individual pair-wise permutation tests (Procrustes distances) between wild-type and enhancer deletion lines revealed significant differences (table S4), with the most pronounced differences observed for Δhs1431 and Δhs746 (both P < 0.0001 compared with wild-type). Differences between wild-type, Δhs1431, and Δhs746 mice were also significant after Bonferroni adjustment for the six pairwise comparisons between groups. The largest magnitude of effect on shape was observed for Δhs1431, followed by an intermediate quantitative effect for Δhs746 (Fig. 7B), whereas possible changes in Δhs586 were not statistically significant after correction for multiple hypothesis testing. These results mirror the magnitude of expression phenotypes, which were most pronounced in Δhs1431, followed by intermediate changes in Δhs746 and only a limited expression phenotype observed in Δhs586 (Fig. 6 and fig. S4). These results show that deletion of enhancers can affect craniofacial morphology.

Fig. 7. Enhancer deletions cause changes of craniofacial morphology. (A) Canonical variate analysis (CVA) of micro-CT data from mice with three different enhancer deletions, compared with wild-type. The 3D morphs show the morphological variation that corresponds to the first three canonical variates. Renderings show CV endpoints 3× expanded so as to improve visualization. (B) Magnitude of shape differences between wild-type and enhancer null mice, based on Procrustes distances (30). Error bars indicate SD of shape differences from resampling Procrustes distances across 10,000 iterations. (C) Wireframe visualization of the first three canonical variates, which are predominantly driven by morphological differences between wild-type mice and Δhs1431, Δhs746, and Δhs586, respectively. CV endpoints are superimposed as red and blue wireframes, respectively.

Panel A

These plots and morphs of adult mouse skulls are evidence that enhancers influence craniofacial shape and that deleting activity of particular enhancers can lead to subtle changes in the shape of adult skulls.

Each of the three enhancer deletions can clearly be seen in separate clusters from the wildtype mice.

WT mice are the negative control.

Panel B

The largest changes in skull shape were observed in the hs1431 deletion mice.

The most subtle changes were observed in hs586 deletion mice.

Panel C

These are wire frame models of mouse skulls and they are presented as further evidence of the influence that enhancer deletions have on skull shape.

Each enhancer deletion causes a distinct set of differences as compared with wild-type morphology. This is evident from the CVA, in which the first three canonical variates (CV1 to CV3) most clearly separate wild-type mice from Δhs1431, Δhs746, and Δhs586, respectively (Fig. 7). Each enhancer deletion produces phenotypic effects that are not confined to a single feature but involve multiple regions of the skull (Fig. 7C and movies S12 to S20). For example, deletion of hs1431 results in an increase in facial length, a relative increase in the width of the anterior neurocranium, and a shortening of the anterior cranial base. In contrast, Δhs746 results in a shortening of the face, a widening of the posterior neurocranium, a narrowing of the palate, and shortening of the cranial base. Although both Δhs1431 and Δhs746 have significant effects on facial morphology in structures derived from regions with enhancer activity at e11.5 and e13.5 (Fig. 6), there are also changes in other parts of the skull. These correlated patterns of change are consistent with numerous studies demonstrating that cranium development is a highly integrated process and that variation of the skull is structured by complex interactions between the growing chondrocranium, neurocranium, and other nearby tissues (52, 53). Regardless of the precise molecular pathways and developmental mechanisms that underlie the morphological changes observed upon deletion of these enhancers, these results demonstrate that distant-acting enhancers contribute to the development of craniofacial shape in mammals. The observation of significant but nonpathological alterations of craniofacial morphology as a result of enhancer deletions supports the notion that enhancers contribute to normal variation in facial shape.

Conclusions

The general shape of the human face and skull, the differences in facial shape between individuals, and the high heritability of facial shape are subjects of broad interest because they have far-reaching implications well beyond basic scientific and biomedical considerations. In this study, we examined the possible impact of distant-acting regulatory sequences on craniofacial morphology. Throughout the genome, we identified several thousand sequences that are likely to be distant-acting enhancers active in vivo during mammalian craniofacial development. Although this epigenomic analysis was performed in the mouse, the vast majority of these enhancer candidate sequences are conserved between mouse and human. Large-scale characterization of more than 200 candidate sequences in transgenic mice showed the versatility of enhancers in orchestrating gene expression during craniofacial development. These observations are consistent with genome-wide analyses of enhancers active in human neural crest cells, as well as studies of regulatory sequences associated with individual members of the neural crest gene regulatory network (23–27). We also demonstrated that deletion of craniofacial enhancers results in nonpathological but measurable changes in craniofacial morphology in mice. Taken together, these data support that enhancers are involved in determining craniofacial shape. Systematic genome-wide studies of normal morphological variation in human populations are beginning to emerge (15–17) and will offer the opportunity to compare in vivo–derived genome-wide maps of craniofacial enhancers identified in this study with variation data in order to gain further mechanistic insight into the molecular underpinnings of human facial shape and variation therein.